The IRAP protein (Insulin-Regulated AminoPeptidase; EC 3.4.11.3) also known as Placental Leucine AminoPeptidase (P-LAP) and Leucine-cystinyl aminopeptidase (L-CAP) is a transmembrane zinc metalloproteinase protein which exists in three isoforms (Swiss-Prot: Q9UIQ6:1, 2 and 3; represented respectively by SEQ ID No: 1 to 3).
When it is inserted into the plasma membrane its extracellular domain can be cleaved and secreted.
The secreted part of an unspecified isoform of the protein assumed to be IRAP was assayed in the blood by an enzymatic method using a non-specific synthetic substrate, L-leucine-nitroanilide in the presence of methionine (Mizutani, S., Yoshino, M., and Oya, M. (1976) Clin Biochem 9(1), 16-18).
Using this method Mizutani, Oya and Tomoda detected a cystinyl-leucine aminopeptidase in the serum of pregnant women, the concentration of which increases during pregnancy (Yamahara, N., Nomura, S., Suzuki, T., Itakura, A., Ito, M., Okamoto, T., Tsujimoto, M., Nakazato, H., and Mizutani, S. (2000) Life Sci 66(15), 1401-1410). This aminopeptidase is essentially of placental origin, and therefore called P-LAP (Tsujimoto, M., Mizutani, S., Adachi, H., Kimura, M., Nakazato, H., and Tomoda, Y. (1992) Arch Biochem Biophys 292(2), 388-392) and corresponds to the secreted domain of the protein.
This enzyme degrades oxytocin (Naruki, M., Mizutani, S., Goto, K., Tsujimoto, M., Nakazato, H., Itakura, A., Mizuno, K., Kurauchi, O., Kikkawa, F., and Tomoda, Y. (1996) Peptides 17(2), 257-261), vasopressin (Wallis, M. G., Lankford, M. F., and Keller, S. R. (2007) Am J Physiol Endocrinol Metab 293(4), E1092-1102), angiotensin II and III (Matsumoto, H., Rogi, T., Yamashiro, K., Kodama, S., Tsuruoka, N., Hattori, A., Takio, K., Mizutani, S., and Tsujimoto, M. (2000) Eur J Biochem 267(1), 46-52) as well as a series of other peptides (Albiston, A. L., Peck, G. R., Yeatman, H. R., Fernando, R., Ye, S., and Chai, S. Y. (2007) Pharmacol Ther 116(3), 417-427). The serous concentrations of this aminopeptidase have never been reported in men or non-pregnant women.
At the time of its cloning, it appeared that P-LAP corresponds to the IRAP protein as well as to the angiotensin IV receptor (Keller, S. R., Scott, H. M., Mastick, C. C., Aebersold, R., and Lienhard, G. E. (1995) J Biol Chem 270(40), 23612-23618; Rogi, T., Tsujimoto, M., Nakazato, H., Mizutani, S., and Tomoda, Y. (1996) J Biol Chem 271(1), 56-61; Albiston, A. L., McDowall, S. G., Matsacos, D., Sim, P., Clune, E., Mustafa, T., Lee, J., Mendelsohn, F. A., Simpson, R. J., Connolly, L. M., and Chai, S. Y. (2001) J Biol Chem 276(52), 48623-48626).
Patent Application WO 2005/038462 describes a reagent for diagnosis and/or prognostic evaluation of carcinoma, comprising an anti-P-LAP polyclonal antibody, obtained by immunization with the whole P-LAP protein. Given the strong homology between the different aminopeptidases, the antibody is probably not specific to IRAP and should also recognize other aminopeptidases. It should not therefore make it possible to diagnose a pathology linked in a precise manner to a modification of the expression or plasmatic concentration of IRAP.
GLUT4 is the glucose transporter which allows the uptake of circulating glucose by the muscles and the adipose tissue in response to insulin. Under unstimulated conditions (basal conditions), GLUT4 is effectively retained inside the cell in intracellular compartments (vesicules), by a still-unknown retention mechanism. In response to insulin stimulation, GLUT4 is transported then inserted into the plasma membrane by increased translocation, thus the allowing the cellular uptake of glucose.
IRAP co-localizes with the glucose transporter GLUT4 and is co-translocated with the latter stoichiometrically (Keller, S. R. (2004) Biol Pharm Bull 27(6), 761-764). This translocation towards the plasma membrane is stimulated by insulin in the same manner as that of GLUT4 (Karylowski, O., Zeigerer, A., Cohen, A., and McGraw, T. E. (2004) Mol Biol Cell 15(2), 870-882; Subtil, A., Lampson, M. A., Keller, S. R., and McGraw, T. E. (2000) J Biol Chem 275(7), 4787-4795).
Moreover, the expression of IRAP conditions the expression of GLUT4 as, in IRAP−/− transgenic animals, the GLUT4 levels are reduced by 50 to 80% (Keller, S. R., Davis, A. C., and Clairmont, K. B. (2002) J Biol Chem 277(20), 17677-17686).
In type 2 diabetics both a reduction of the expression and the translocation of GLUT4 towards the plasma membrane in the muscle and the adipose tissue is found (Kahn, B. B. (1992) J Clin Invest 89(5), 1367-1374).
Similarly and although the cellular levels of IRAP are not modified, its translocation is also reduced in the muscle and the adipose tissue of type 2 diabetics (Garvey, W. T., Maianu, L., Zhu, J. H., Brechtel-Hook, G., Wallace, P., and Baron, A. D. (1998) J Clin Invest 101(11), 2377-2386; Maianu, L., Keller, S. R., and Garvey, W. T. (2001) J Clin Endocrinol Metab 86(11), 5450-5456).
Moreover, a relationship has been demonstrated between the IRAP protein and the development of chemoresistance to anticancer drugs (Kondo C. et al., Int J. Cancer, 118, 1390-1394, 2006). According to this article, IRAP in fact reduces sensitivity to anticancer drugs by inhibiting the expression of the apoptosis-induction factor and increases expression of the apoptosis-inhibition factor.
The extracellular domain of IRAP is cleaved by metalloproteases probably belonging to the ADAM family including ADAM9 (SwissProt Q13443) and ADAM12 (SwissProt O43184) (Ito, N., Nomura, S., Iwase, A., Ito, T., Kikkawa, F., Tsujimoto, M., Ishiura, S., and Mizutani, S. (2004) Biochem Biophys Res Commun 314(4), 1008-1013) and released into the blood circulation. ADAM9 (MDC9) is expressed in different tissues including the skeletal muscle and adipose tissue (Hotoda, N., Koike, H., Sasagawa, N., and Ishiura, S. (2002) Biochem Biophys Res Commun 293(2), 800-805) and ADAM12 is essentially expressed in the muscle. Several other members of this family are also expressed in the muscle and adipose tissue.
At present, the only relationships described between the concentration of the extracellular domain of IRAP in a biological medium and a pathology concern severe preeclampsia as well as the risk of premature labour. In these two pathologies the circulating P-LAP concentrations are less than those observed in the control women of equivalent gestational age. However, the methods described in the prior art are based on the enzymatic assay, using a non-specific substrate, of the extracellular domain of IRAP.
There is therefore a real need to provide a reliable and specific method making it possible to assay the extracellular domain of circulating IRAP.